The present invention relates generally to microanalytical devices for use in analysis of complex molecules and biomolecules, and more specifically, to microanalytical devices containing a membrane for biomolecular identification.
There is a rapidly growing awareness of the importance of biomolecular identification in the discovery of medically important proteins and the genes from which they derive, e.g., proteomics and genomics. For example, many of the best-selling drugs today either are proteins or act by targeting proteins. In addition, many molecular markers of disease, the basis of diagnostics, are peptidic or nucleotidic sequences. It is evident that biomolecular identification techniques such as proteomics and genomics have major implications for pharmaceutical research and development.
Biomolecular identification often involves the analysis of large complex biomolecules, typically peptidic biomolecules such as proteins in the case of proteomics, by breakdown thereof into simpler component substances. Two-dimensional electrophoresis technology forms a current basis of the expanding field of proteomics in large part because of the high resolution obtainable from multidimensional separation. For example, two-dimensional electrophoresis has been widely used to separate hundreds to thousands of proteins in a single analysis, in order to determine the protein composition of biological samples such as blood plasma, tissues, cultured cells, etc. Such two-dimensional procedures may involve sequential separations by isoelectric focusing and slab gel electrophoresis followed by mass spectrometry. However, this technology suffers from its slow and labor-intensive nature. Thus, automation of the procedure is desired for scale-up of efforts to build proteome databases comprising all the proteins of complex organisms such as human beings. For instance, U.S. Pat. No. 5,993,627 to Anderson et al. discloses an automatic system for two-dimensional electrophoresis. However, this system is complex in design and is likely to require a large amount of sample for proper functioning. Not surprisingly, two-dimensional electrophoresis is still ordinarily performed by hand or on a bench scale level.
Other automated approaches may also enhance the speed of analysis. One such approach involves the use of devices capable of carrying out analysis such as multidimensional liquid chromatography-mass spectrometry. For example, U.S. Pat. No. 5,705,813 to Apffel et al. describes an integrated planar liquid handling system for matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The patent discloses that a reservoir for receiving fluid substances may be interconnected by a microchannel to a MALDI ionization surface, wherein the microchannel comprises a separation region. The separation region may be used for chromatographic-type separations. As another example, U.S. Pat. No. 5,716,825 to Hancock et al. describes an integrated nucleic acid analysis system for MALDI-TOF mass spectrometry. However, the advantages obtained from such high speed analysis are offset by lack of resolution and specificity.
These automated approaches represent recent progress in microfabricated devices used, e.g., as chemical analysis tools or clinical diagnostic tools. Their small size allows for the analysis of minute quantities of sample, which is an advantage where the sample is expensive or difficult to obtain. See, e.g., U.S. Pat. No. 5,500,071 to Kaltenbach et al., U.S. Pat. No. 5,571,410 to Swedberg et al., and U.S. Pat. No. 5,645,702 to Witt et al. Sample preparation, separation and detection compartments have been proposed to be integrated on such devices. Because such microfabricated devices have a relatively simple construction, they are generally inexpensive to manufacture. Nevertheless, the production of such devices present various challenges. For example, the flow characteristics of fluids in the small flow channels of a microfabricated device may differ from the flow characteristics of fluids in larger devices, as surface effects come to predominate and regions of bulk flow become proportionately less. Thus, motive force means for producing a motive force that moves analytes and fluids may have to be incorporated into such microanalytical devices. This may involve forming motive force means such as electrodes on such microanalytical devices which may add to the cost of the device. Moreover, microanalytical devices have never been constructed that have the high resolution capabilities of two-dimensional electrophoresis as a whole.
Another approach in controlling flow characteristics in fluids is to employ membranes having structures with field responsive permeation control. Such structures have been described in a number of patents and publications and may be incorporated into microporous membranes. U.S. Pat. No. 5,556,528 to Bohn et al., for example, describes structures with field response permeation control. A support is interposed in a passageway between a fluid source with target molecules therein and a fluid reservoir, wherein the support permits target molecules to diffuse therethrough. The surface of the support carries at least one monomolecular layer which forms, in part or in whole, an active control structure. The active control structure is an admixture of molecular species, a majority of which has a large ground state dipole moment. The monomolecular layers have closely packed, dipolar molecules that are substantially aligned along their long axes. When an electric field is applied or removed, the permeability of the active control structure is altered. The target molecules are captured or dispensed according to the permeability value. Similarly, International Publication No. WO 99/65945 describes force-regulated molecular recognition switches for controlling binding and release of a ligand to a device containing such a switch. However, such structures have not been used to chemically modify, or more specifically, to cleave, digest, or otherwise segment analyte biomolecules into separate components.
Accordingly, there is a need for a device that requires only small volumes of sample fluid for biomolecular identification applications, particularly proteomics and genomics. In addition, there is a need to maintain high resolution with high speed analysis by allowing parallel chemical alteration, e.g., proteolytic processing, of samples in a microfluidic system.
Accordingly, it is an object of the present invention to overcome the above-mentioned disadvantages of the prior art by providing a microanalytical device containing a membrane for biomolecular identification.
It is another object of the invention to provide such a device that allows biomolecular identification to be carried out with speed, high resolution and specificity.
It is still another object of the invention to provide such a microanalytical device for biomolecular identification through spatial resolution and digestion.
It is a further object of the invention to provide a method for biomolecular identification using such a microanalytical device.
Additional objects, advantages and novel features of the invention will be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
In a general aspect, then, the present invention relates to a microanalytical device for analyzing a fluid sample containing at least one analyte molecule. The microanalytical device is constructed from a substrate, a cover plate and a membrane. The substrate has a substantially planar substrate surface and a substrate microchannel formed thereon. The cover plate has a substantially planar cover plate surface and a cover plate microchannel formed thereon. The cover plate surface is arranged over the substrate surface, and the membrane is interposed between the substrate and the cover plate. The membrane has at least one pore sized to allow passage of the analyte molecule from the substrate microchannel to the cover plate microchannel. An analyte altering moiety is attached to an interior surface of the at least one pore and is capable of chemically altering the analyte molecule.
In another general aspect, the invention relates to a method for chemically altering and transporting an analyte molecule in a fluid. The method involves providing a substrate having a substantially planar substrate surface, the substrate having a substrate microchannel formed in the substrate surface. The analyte molecule is transported from the substrate microchannel in a non-parallel direction with respect to the substrate surface into a pore of a membrane. Once inside the pore, the analyte molecule is chemically altered using a moiety attached to an interior surface of the pore to form an altered molecule. The altered molecule is collected from the pore on a cover plate microchannel formed on a substantially planar cover plate surface of a cover plate, wherein the substantially planar cover plate surface is in opposing relation to the substantially planar substrate surface.
In a further general aspect, the invention relates to a method for identifying a plurality of biomolecules. First, a substrate having a substantially planar substrate surface is provided having a substrate microchannel formed therein, and the to biomolecules are spatially resolved along the substrate microchannel. Then the biomolecules are transported from the substrate microchannel in a non-parallel direction with respect to the substrate surface into pores of a membrane while preserving the spatial resolution of the biomolecules. There, the biomolecules are digested to form biomolecular components. The biomolecular components, still spatially resolved, are then collected from the pores on a cover plate microchannel channel formed on a substantially planar cover plate surface of a cover plate, wherein the substantially cover plate surface is in opposing relation to the substantially planar substrate surface. Finally, the biomolecular components are spatially resolved in the cover plate microchannel.